Poliovirus, one of the human picornaviruses of the enterovirus subgroup, has been extensively studied because it is the causative agent for serious human disease. Because of these studies, it is now known that the virion of poliovirus consists of a small icosahedron, 25-30 nm in diameter, composed of four polypeptides, which are designated VP1, VP2, VP3 and VP4. A single strand of infectious positive-stranded RNA of molecular weight 2.7.times.10.sup.6 daltons is enclosed within this protein coat. This size is equivalent to approximately 7500 bases, which can code for about 2500 amino acids.
The first significant breakthrough in research directed to limiting the spread of epidemics of poliovirus was attained by the development of a killed virus vaccine for poliovirus by Jonas Salk in 1953. See Salk, J. E. with the collaboration of Bennett, B. L., Lewis, L. J., Ward, E. N., and Youngner, J. S., "Studies in Human Subjects on Active Immunization Against Poliomyelitis. I. A Preliminary Report of Experiments in Progess.", J. Am. Med. Assoc. 151 1081-1098 (1953). Preparation of the Salk killed virus vaccine was achieved by producing large amounts of virus in cell culture and subsequently treating the infected tissure culture fluid with formalin to inactivate the virus particles.
More recently, live attenuated polio vaccines have been developed. See Koprowski, H., Jervis, G. A. and Norton, T. W., "Immune Response in Human Volunteers Upon Oral Administration of A Rodent Adapted Strain of Poliomyelitis Virus", Am. J. Hyg. 55 108-126 (1952); and Sabin, A., "Properties of Attenuated Poliovirus and Their Behavior in Human Beings", Spec. Publ. N. Y. Acad. Sci. 5, 113-127 (1957). Live attenuated polio vaccine proved to be equal to or more effective than killed polio virus vaccine in reducing the incidence of poliomyelitis and possessed substantial practical and theoretical advantages. Such vaccines were simple to administer and entered through the normal routes, and were found to induce higher levels of circulating antibody as well as the production of IgA antibody from local exocrine cells.
Despite the marked success achieved by prior polio vaccines, there remains even today a strong desire to develop new vaccines which would be easier to manufacture that the Salk vaccine and eliminate or substantially diminish the possibility of attenuated viruses regaining virulence and therefore becoming capable of infecting recipients.
In particular, there has been much research devoted towards determining whether subviral poliovirus components, particularly any of the capsid polypeptides VP1, VP2, VP3 or VP4, might be capable themselves of producing neutralizing antibodies upon injection into a host. If such subviral components could produce neutralizing antibodies, there would be great advantages in forming vaccines from such subviral components since there would be no possibility that such components could be infectious and since such components could most likely be produced in commercial quantities by cloning the gene sequence of such components in a recombinant cDNA vector.
Support for the belief that individual capsid polypeptides of poliovirus might be capable of generating neutralizing antibodies was initially based upon research with isolated capsid proteins of foot-and-mouth disease virus. In this work, one of the capsid proteins of foot and mouth disease virus, termed VP3 (sometimes VP.sub.t or even VP1), was isolated and injected into swine and guinea pigs. It was found that the foot-and-mouth virus polypeptide VP3 was capable of initiated neutralizing antibody production in both. On the other hand, neutralizing antibodies were not produced in response to immunization with VP1 or VP2 polypeptides isolated from the foot and mouth disease virus capsid. See Bachrach, H. L., Moore, D. M., McKercher, P. D. and Polatnick, J., "Immune and Antibody Responses to an Isolated Capsid Protein of Foot-and-Mouth Disease Virus", J. Immunol. 115, No. 6, pp 1636-41 (1975). It was also demonstrated that mengovirus, another member of the aphthovirus subgroup, also produced neutralizing antibody in animals, Lund, G.A., Ziola, B.R., Salmi, A. and Scroba, D.G., "Structure of the Mengo Virion. V. Distribution of the Capsid Polypeptides With Respect to the Surface of the Virus Particle," Virology 78, 35-44 (1977).
Despite this work with foot-and-mouth virus and mengovirus, attempts to produce neutralizing antibodies from capsid polypeptides of the poliovirus met with no success or were inconclusive. For example, Meloen et al. compared the individual capsid polypeptides of foot-and-mouth and polio viruses and found that none of the individual polypeptides from poliovirus produced antisera which neutralized the virus. See Meloen, R. H., Rowlands, D. J. and Brown, F., "Comparison of the Antibodies Elicited by the Individual Structural Polypeptides of Foot-and-Mouth Disease and Polioviruses", J. Gen Virol. 45, 761-3 (1979).
Icenogle et al. prepared a neutralizing monoclonal antibody against Type 1 poliovirus and attempted to determine the affinity of this monoclonal antibody against various subviral particles. The monoclonal antibody had a much higher affinity for the 14S subunit than for similar subunits lacking the VP1 polypeptide, leading the authors to conclude that the VP1 polypeptide had an important role in the production of neutralizing antibodies. However, the monoclonal antibody had an almost insignificant affinity for the individual polypeptides. See FIG. 2, Icenogle, J., Gilbert, S. F., Grieves, J., Anderegg, J. and Rueckert, R., "A Neutralizing Monoclonal Antibody Against Poliovirus and Its Reaction With Related Antigens", Virology 115, 211-15 (1981). Similar reasoning had earlier led Breindl to conclude that the VP4 polypeptide of poliovirus contained the reactive antigenic determinants. See Breindl, M., "VP4, The D-Reactive Part of Poliovirus," Virology 46; 962-64 (1971). VP4, however, is not present on the surface of the poliovirus capsid and is not presently believed to be capable of producing neutralizing antibodies today.
The most recent report in the literature concerning efforts to produce neutralizing antibodies with poliovirus capsid polypeptides presents the results of Blondel et al. at the French Academy of Science. See Blondel, B., Crainic, R. and Horodniceanu, F., "Le Polypeptide Structural VP1 du Poliovirus Type 1 Induit Des Anticorps Neutralisants", C. R. Acad. Sc. Paris, t294 (Serie III-91(1982). In this report, the researchers state that the poliovirus polypeptide VP1 produced neutralizing antibodies in one of two rabbits they immunized. Data were not reported, however, to rule out the possibility that the VP1 generated antisera recognized antigenic determinants present on the VP2 or VP3 proteins.